Functionalized Mesoporous Silica via an Aminosilane Surfactant Ion Exchange Reaction: Controlled Scaffold Design and Nitric Oxide Release

ABSTRACT

Nitric oxide-releasing mesoporous silica nanoparticles (MSNs) were prepared using an aminosilane-template surfactant ion exchange reaction. Initially, bare silica particles were synthesized under basic conditions in the presence of cetyltrimethylammonium bromide (CTAB). These particles were functionalized with nitric oxide (NO) donor precursors via the addition of aminosilane directly to the particle sol, and a commensurate ion exchange reaction between the cationic aminosilanes and CTAB. N-diazeniumdiolate NO donors were formed at the secondary amines to yield NO-releasing silica MSNs. Tuning of the ion exchange-based MSN modification approach allowed for the preparation of monodisperse particles ranging from 30 to 1100 nm. Regardless of size, the MSNs stored appreciable levels of NO (0.4-3.5 μmol/mg) with tunable NO-release durations (1-33 h) dependent on the aminosilane modification. The range of MSN sizes and NO release demonstrate the versatility of this strategy.

This invention claims priority under 35 USC 365, 35 USC 371, and 35 USC119(e) to U.S. Provisional Application No. 62/249,428 filed Nov. 2,2015, the entire contents of which are incorporated by reference in itsentirety for all purposes.

This invention was supported at least in part by National ScienceFoundation Grant DMR1104892. Thus, the Federal Government has rights inthis invention.

BACKGROUND OF THE INVENTION

Nitric oxide (NO), an endogenous diatomic free radical, mediatesmultiple physiological processes including angiogenesis, blood pressureregulation, wound healing, and the immune response. In vivo, nitricoxide synthase (NOS) enzymes generate NO at concentrations (nM-μM) andkinetics dependent on the enzyme location and purpose. For example, lowconcentrations of NO generated via calcium-dependent endothelial andneuronal NOS regulate neovascularization and serve roles inneurotransmission. Activation of the inducible NOS isoform byimmunological stimuli (e.g., lipopolysaccharide, interferon-γ) causessustained NO release at high concentrations to eradicate foreignpathogens as part of the innate immune response. The multifaceted rolesof endogenous NO are attributable to precise spatiotemporal NO releaseby cells expressing the NOS enzymes. In addition, NO's short biologicallifetime (seconds) restricts its action to <0.5 mm from the point ofgeneration.

Due to NO's overwhelming presence in physiology, the administration ofexogenous NO gas represents a potential therapy for many diseases. Asignificant body of research has focused on the development of donorsthat store and release NO under specific chemical conditions in order toaddress the concentration-dependent behavior of NO and avoid challengesassociated with the administration of NO directly, such as the need fora pressurized gas cylinder and NO's rapid reaction in biological media.For example, N-diazeniumdiolate NO donors, formed by the reaction ofgaseous NO with secondary amines, spontaneously release NO inphysiological buffer upon reaction with hydronium ions. This class ofmolecules has accordingly received attention for biological applicationsbecause the breakdown of the NO donor and concomitant NO release occursat rates dependent on pH, temperature, and the chemical structure of theprecursor molecule used for N-diazeniumdiolate formation.

The potential utility of the N-diazeniumdiolate functional grouporiginally inspired research on low molecular weight NO donors.Unfortunately, limited NO capacity and duration generally preclude theuse of these small molecule NO donors for therapeutic applications. Toenhance NO storage and exert additional control over NO release, muchwork has focused on the synthesis of N-diazeniumdiolate-modifiedmacromolecular NO-delivery scaffolds, including chitosanoligosaccharides, dendrimers, gold clusters, and silica nanoparticles.With respect to silica, surface grafting, co-condensation, andwater-in-oil microemulsion methods have been used to prepareN-diazeniumdiolate-functionalized particles. Silica is attractive as anNO-release scaffold as it is well tolerated (i.e., nontoxic) and readilyimplemented as a drug delivery vehicle. For example, NO donor-modifiedsilica particles have served as reinforcing fillers for NO-releasingpolymeric coatings to promote angiogenesis and wound healing. Suchmaterials have also proven effective as antimicrobial abrasives that maybe integrated with oral hygiene technologies.

Despite their value as potential therapeutics, current strategies forsynthesizing NO-releasing silica nanoparticles remain limited bychallenges associated with altering the physical properties of theparticles and the NO release independent of one another. The use ofmesoporous silica represents an attractive macromolecular scaffold forenhancing NO storage and release because of the inherently greater andmodifiable surface area (>1,000 m²/g) relative to previous nonporoussilica systems. Control over pore formation and the silica mesophase isachieved via the synthesis of the nanoparticles around an orderedsurfactant aggregate, generally an alkyltrimethylammonium salt, whichserves as the structure-directing agent (SDA). Covalent attachment ofsecondary-amine containing silanes (i.e., NO donor precursors) tomesoporous silica is typically carried out by direct incorporation ofthe aminosilane into the particle backbone via co-condensation orpost-synthetically through surface grafting. In the co-condensationapproach, coulombic repulsion between the cationic surfactant moleculesand the protonated backbone amines destabilizes the template, resultingin materials with irregular morphology, even at low aminosilaneconcentrations. Post-synthetic surface grafting (after extracting theSDA) is generally the preferred method for functionalizing mesoporoussilica, albeit at the cost of increased synthetic burden. Moreover, thegrafting process requires a nonpolar aprotic solvent to avoidirreversible water-induced particle agglomeration, heterogeneous aminedistribution, and hatch-to-batch reproducibility.

The synthesis of NO-releasing nanoparticles has been previouslyreported, but without autonomous control over particle size, NO-releasekinetics, and NO storage. Generally, total NO storage for silica-basedmaterials is limited to <0.40 μmol/mg due to low aminosilaneincorporation. Limited NO storage often is further compounded by a lackof morphological control and poor synthesis yields.

It is with these shortcomings in mind that the present invention wasdeveloped.

BRIEF SUMMARY OF THE INVENTION

An ion exchange reaction with C₍₅₋₁₅₎alkyltrimethylammonium halides canbe used to prepare a diverse selection of monodisperse NO-releasingamine-functionalized mesoporous silica nanoparticles (MSNs) by directaddition of the aminosilane to the particle sol. It is contemplated andtherefore within the scope of the present invention that a similar ionexchange approach would be feasible using cetyltrimethylammoniumchloride (i.e., an SDA with another halide counterion). Other salts witha range of cation structures are also appropriate for the presentinvention. Some examples of these include SDAs with chemical structuresthat accompany the list given below:

-   -   a. Linear alkylammonium salts with molecular structures        [C_(n)H_(2n+1)(CH₃)₃N]⁺ Br⁻ or [C_(n)H_(2n+1)(C₂H₅)₃N]⁺ Br⁻        where n=8, 10, 12, 14, 16, 18, 20, 22.    -   b. Germinal salts with general molecular structures        [C_(n)H_(2n+1)(CH₃)₂N—(CH₂)₈—N(CH₃)₂—C_(m)H_(2m+1)]⁺ Br⁻ where        n=m=12, 14, 16, 18, 20, 22 and s=2−12.    -   c. Divalent surfactants with general molecular structures        [C_(n)H_(2n+1)(CH₃)₂N—C_(m)H_(2m+1)(CH₃)₃N]²⁺ where n+m=8, 10,        12, 14, 16, 18, 20, 22.

For example, cetyltrimethylammonium bromide (CTAB) has been used toprepare a diverse selection of monodisperse NO-releasingamine-functionalized mesoporous silica nanoparticles (MSNs) by directaddition of the aminosilane to the particle sol. The surface- andpore-bound secondary amines may then be converted to N-diazeniumdiolatemoieties to yield the NO-releasing MSNs. The relationships betweenNO-release kinetics and particle structure (i.e., pore organization,aminosilane modification) have been elucidated via detailedphysicochemical analysis of the MSNs.

Ion exchange between cationic organosilanes and alkyltrimethylammoniumSDAs represents a new MSN functionalization approach.

Nitric oxide-releasing mesoporous silica nanoparticles with a range ofsizes (30, 150, 450, and 1.100 nm) were successfully prepared using anaminosilane-CTAB ion exchange approach. The resulting MSNs werewell-defined and exhibited a large degree of surface modification, whichtranslated to competitive NO storage with other macromolecular NO donors(e.g., MOFs, RSNOs). Particle NO storage and release kinetics weredependent on both the structure of the pores and the identity of theprecursor aminosilane. This invention is the first to detail thedependence of NO-release kinetics on the architectural properties ofmesoporous silica.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows nitrogen adsorption and desorption isotherms for bare (A)1100; (B) 450; (C) 150; and (D) 30 nm MSNs. The estimated pore widthdistributions calculated via BJH analysis of the adsorption branch arepresented in (E) for the 1100 (black), 450 (blue), 150 (red), and 30 mm(green) particles.

FIG. 2 shows transmission electron micrographs of (A) 1100; (B) 450; (C)150; and (D) 30 nm AEAP3-modified mesoporous silica nanoparticles.

FIG. 3 shows solid-state CP/MAS ²⁹Si NMR spectra of (A) 1100; (B) 450;(C) 150; and (D) 30 nm AEAP3-modified particles.

FIG. 4 shows small-angle X-ray scattering profiles for (A) 1100; (B)450; (C) 150; and (D) 30 nm MSNs.

FIG. 5 shows a two-step synthetic scheme to generate MSNfunctionalization with aminosilanes. The first part involvesbase-catalyzed synthesis of bare mesoporous silica scaffold. The secondpart of the scheme shows a proposed mechanism for MSN functionalizationwith aminosilanes. Positively charged aminosilanes undergo ion exchangewith the template surfactant to stabilize anionic silanol speciesanchored to the mesopore walls.

FIG. 6 shows transmission electron micrographs of 150 nm MSNs modifiedwith (A) APTES; (B) BTMS; and (C) MPTMS. Particles in (A) exhibit smoothmorphology, while agglomeration is observed in (B) and (C).

FIG. 7 shows scanning electron micrographs of 1100 nm AEAP3-modifiedparticles with reactant AEAP3 concentrations of (A) 11.47 mM and (B)14.34 mM. While the particles in (A) exhibited smooth morphology,undesirable particle agglomeration occurred at higher AEAP3concentrations.

FIG. 8 shows small-angle X-ray scattering profile for AEAP3-modified (A)1100 nm; (B) 450 nm; (C) 150 nm; and (D) 30 nm MSNs.

FIG. 9 shows real-time NO-release profiles for 30 nm MAP3/NO (black),AHAP3/NO (red), AEAP3/NG (green), and DET3/NO (blue) particles after ˜20min in PBS (pH 7.4) at 37° C.

DETAILED DESCRIPTION OF THE INVENTION

Nitric oxide-releasing silica nanoparticles have demonstrated promise asantimicrobials, but are traditionally limited in terms of theiruncontrollable physical properties (e.g., sizes, low nitric oxidestorage, and poor synthesis yields. In an embodiment, the presentinvention relates to a new preparative strategy for nitricoxide-releasing silica nanoparticles that exploits ion exchangereactions between aminosilanes used for nanoparticle functionalizationand the cationic template surfactant that controls nanoparticleformation. When compared with previous synthesis protocols, theefficiency of this process allows for autonomous control over particlephysicochemical properties including nitric oxide storage, nitricoxide-release kindles, and particle size. Synthesis yields as high as0.8 grams also represent a significant advantage over other strategies(0.01-0.1 grams) and the flexibility of this process for particlemodification with several functional cationic organosilanes wasdemonstrated. Prior to this invention, mesoporous silica materials couldbe functionalized through either co-condensation or surface grafting.Modification via ion exchange reactions holds the following advantagesover pre-existing methods: 1) Consistent material morphology regardlessof aminosilane incorporation (not possible with co-condensation,difficult and irreproducible with surface grafting) 2) One-pot synthesisof amine-functionalized materials (not possible with surface grafting)3) Insensitive to ambient humidity (not possible with surface grafting)4) Streamlined material synthesis and aminosilane incorporation isreproducible.

Additionally, other disadvantages of the prior surface grafting methodinclude low nitrogen incorporation and NO storage (see table 8 fortypical values). Moreover, surface grafting has a water sensitive stepthat often, results in irreproducible percentage of nitrogenincorporation and uneven amine special distribution. Co-condensationdrawbacks include generating core amines that tend to be inaccessiblefor further chemical modification and no to little morphologicalcontrol.

Prior art synthesis of NO-releasing nanoparticles suffers from thedrawback of lacking autonomous control over particle size, NO-releasekinetics, and NO storage. Generally, total NO storage for silica-basedmaterials is limited to <0.40 μmol/mg due to low aminosilaneincorporation. Limited NO storage often is further compounded by a lackof morphological control and poor synthesis yields. Accordingly, thepresent invention relates to a new method of synthesis whereinmesoporous silica was selected as a new scaffold in an attempt to exertgreater control over particle NO-release properties. Mesoporous silicananoparticles were prepared via a supramolecular liquid-crystaltemplating approach. Cationic, amphophilic CTAB aggregates were used asthe structure-directing agent for particle synthesis. The synthesis offour different sized MSNs was achieved using tetraethylorthosilicate(TEOS) by altering the reaction temperature and reactant concentrations(See Table 1). For example, by employing lower temperatures (on theorder of 20-25° C.) lesser amounts of water (on the order of 25 M) and ahigher ratio of amine to alkyltrimethylammonium halide, one is able toattain larger particle sizes, whereas higher temperatures (on the orderof 65-70° C.), higher water concentration (on the order of 55 M) and asmaller ratio of amine to alkyltrimethylammonium halide allows one toachieve smaller particle sizes. Thus, on the whole, to generate theparticle sizes of the present invention, the temperature ranged fromabout 20-70° C., the water concentration ranged from about 25-55 M, theamine concentration ranged from about 0.25 M to about 0.55 M, and thealkyltrimethylammonium halide ranged from about 2 mM to 5.5 mM.

Surfactant was removed by ion exchange in ethanolic hydrochloric acid(HCl) to yield the bare mesoporous scaffold. While other methods (e.g.,calcination) have been used for CTAB removal, irreversible particleagglomeration often results. Surfactant removal from the MSNs afteragitation in HCl was evaluated using elemental analysis. The measurednitrogen wt % for the bare particles was <0.2% in all cases (indicatingcomplete CTAB removal), with the exception of the 150 nm system(˜1.11%). The significant nitrogen content was attributed to trappedammonia, since the low carbon content (5.48±1.00%) did not reflect thepresence of CTAB (˜80.3% carbon by mass).

TABLE 1 Synthesis conditions and nitrogen physisorption data for MSNs ofvarying size.^(a) Particle [H₂O] [NH₃] [CTAB] Reaction Specific SurfacePore Width Nitrogen Size (M) (M) (nM) Volume (mL) Temperature (° C.)Area (m² g⁻¹)^(b) (Å)^(c) wt %^(d)  30 nm 54.5 0.267 5.30 150 68 ± 11290 ± 90 23.6 ± 2.3 ≤0.01 150 nm 39.4 0.267 5.30 150 38 ± 1 1170 ± 8021.9 ± 0.6 1.11 ± 0.12 450 nm 35.0 0.267 5.30 150 23 ± 1  1280 ± 12020.4 ± 0.2 0.13 ± 0.06 1100 nm  25.5 0.521 2.20 350 23 ± 1 1170 ± 7019.5 ± 0.3 ≤0.01 ^(a)Error bars represent standard deviation for n ≥3separate syntheses. ^(b) Determined by BET analysis of the nitrogensorption isotherms (0.05 ≤ p/p⁰ ≤ 0.20). ^(c)Calculated via BJH analysisof the nitrogen adsorption isotherm (p/p⁰ ≤0.60). ^(d)Nitrogon wt %measured by elemental analysis.

The surface areas and pore sizes of the unmodified MSNs were calculatedfrom the corresponding nitrogen sorption isotherms (FIG. 1). Each of thephysisorption isotherms exhibited steep inflections at ˜0.2-0.4 p/p⁰and >0.8 p/p⁰ corresponding to capillary condensation of nitrogen in theparticle mesopores and inter-particle volumes, respectively. Allisotherms were classified as Type IV isotherms without hysteresisaccording to the conventions adopted by the International Union of Pureand Applied Chemistry (IUPAC). Nitrogen gas adsorption/desorption onCTAB-templated mesoporous silica has consistently yielded similarresults. Importantly, MSN surface areas calculated using theBrunauer-Emmett-Teller (BET) method exceeded 1000 m²/g in all cases(Table 1) regardless of particle size. Pore sizes were evaluated usingBarrett-Joyner-Halenda (BJH) analysis of a portion of the nitrogenadsorption branch (see FIG. 1e ) and yielded calculated pore widths inthe range of 19.5-23.6 Å, which are comparable to those reported in theliterature.

The particles were modified with secondary amines by direct organosilaneaddition to the reaction solution following completion of the particlesynthesis reaction (<2 h as determined by dynamic light scattering).Exemplary organosilanes include cationic organosilanes such asN-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3),N-[3-(trimeth-oxysilyl) propyl]diethylenetriamine (DET3),N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3) andmethylaminopropyl-trimethoxysilane (MAP3), Other cationic organosilanesthat might potentially be used in the present invention include one ormore of N-(2-aminoethyl)aminopropyltrimethoxysilane,N-methylaminopropyltrimethoxysilane,N-(6-aminohexyl)aminopropyltrimethoxysilane,N-(6-aminohexyl)aminomethyltrimethoxysilane,3-aminopropyltrimethoxysilane,3-(trimethoxysilylpropyl)diethylenetriamine,N-(2-aminoethyl)aminoundecyltrimethoxysilane,aminoethylaminomethyl)phenethyltrimethoxysilane, and mixtures thereof.

Residual surfactant SDA was removed in a subsequent step, similar tounmodified particles. The aminosilaneN-(2-aminoethyl)-3-aminopropyltrimethoxysilane, (AEAP3) was selected tooptimize this process, initially using the largest (1100 nm) particles.Generally, the ion exchange process can be optimized by evaluatingnitrogen incorporation (measured using an elemental analyzer) withrespect to the aminosilane concentration. Additionally, the pH of theprecursor solution (i.e., the particle synthesis solution) may need tobe altered to ensure protonation of the aminosilane reagent.

As expected, elevated nitrogen compositions and lower specific surfaceareas were observed upon increasing the concentration of AEAP3 in thereaction (Table 2). At the highest AEAP3 concentration presented inTable 2 (11.5 mM), particles retained excellent sphericity andmorphology (FIG. 2a ) as indicated by transmission electron microscopy(TEM). At this concentration, a maximum nitrogen content of 4.87±0.04 wt%, was measured, suggesting incorporation of AEAP3 to the MSNs ratherthan the formation of new, discrete AEAP3-based particles. Undesirableparticle agglomeration was routinely noted at greater AEAP3concentrations (>14.3 mM). Inter-particle bridging was occurring atthese higher concentrations, revealing a practical maximum in theattainable aminosilane incorporation (See FIG. S1). Pore size analysisof the nitrogen adsorption isotherms indicated a clear decrease inmesopore volume with increasing AEAP3 concentration, while the porewidth remained invariable (p>0.50). At AEAP3 concentrations at orexceeding 5.7 mM, the gas sorption isotherm abruptly transitioned from atype IV to a type I isotherm, consistent with bound organic groups onthe silica network. Taken together, these data indicate AEAP3infiltration of the particle mesopores at AEAP3 concentrations <11.5 mM.

TABLE 2 Characterization of AEAP3-modified 1100 nm mesoporous silicaparticles as a function of reaction aminosilane concentration.^(a)Specific Surface Cumulative Pore Pore [AEAP3] Area Volume Width Nitrogen(mM) (m² g⁻¹)^(b) (cm³ g⁻¹)^(c) (Å)^(c) wt %^(d) 0 1200 ± 70  0.47 ±0.09 19.5 ± 0.3 ≤0.01 1.4 790 ± 60  0.13 ± 0.02 19.4 ± 0.7 2.41 ± 0.252.9 520 ± 130 0.05 ± 0.01 20.0 ± 0.7 3.38 ± 0.41 5.7 5 ± 1 0.01 ± 0.0020.7 ± 2.0 4.38 ± 0.33 11.5 3 ± 1 0.00 ± 0.00 N.D.

4.87 ± 0.04 ^(a)Error bars represent standard deviation for n ≥ 3separate syntheses. ^(b)Determined by BET anaylysis of the nitrogensorption isotherms (0.05 ≤ p/p⁰ ≤ 0.20). ^(c)Calculated via BJH analysisof the nitrogen adsorption isotherm (p/p⁰ ≤ 0.60). ^(d)Nitrogen wt %measured by elemental analysis.

Pore width could not be calculated from the adsorption isotherm.

indicates data missing or illegible when filed

Based on the results for the 1100 nm particles, the optimalTEOS:aminosilane molar ratio of 1.56:1.00 was used to synthesize smallerAEAP3 particles. The TEOS:aminosilane ratio that was tested was from1.26:1.00 to 12.84:1.00. Regardless of the intended size, this approachresulted in well-defined nanomaterials (FIGS. 2b-d ). Dynamic lightscattering (DLS) analysis of aqueous MSN dispersions (Table 3) supportedTEM observations. The low observed polydispersity indices (PDIs; 0.12,0.02, and 0.04 for the 30, 150, and 450 nm particles, respectively)affirmed narrow particle size distributions. In a separate embodiment,the four particle systems with, nominal diameters of 30, 150, 450, and1100 nm were prepared with PDIs of 0.1.7, 0.05, 0.02, and ˜0.1,respectively. The DLS/TEM data also provided evidence for covalentbonding of aminosilanes to the particle surface, rather than theformation of discrete entities and likely large agglomerates. Elementalanalysis confirmed aminosilane incorporation (>4.50 % N) for eachparticle system, with the 150 nm MSNs exhibiting the greatest nitrogencomposition (5.91% N). Of note, the increased nitrogen observed for the150 nm MSNs is likely due to the large amount of nitrogen in the bare150 nm MSNs (1.11±0.12% N) and did not reflect improved aminosilaneincorporation.

TABLE 3 Physicochemical characterization of AEAP3-fonctionalized MSNs ofvarying size.

Geometric Z-average Specific Surface Diameter Size Area Pore Width(nm)^(b) (nm)^(c) PDI^(c) Nitrogen wt %^(d) (m² g⁻¹)^(e) (Å)^(f) 36 ± 874 ± 6 0.12 ± 0.06 4.65 ± 0.19 210 ± 40  25.1 ± 1.1 149 ± 13 223 ± 170.02 ± 0.01 5.91 ± 0.13 69 ± 13 24.8 ± 0.6 450 ± 50 564 ± 66 0.04 ± 0.025.07 ± 0.10 68 ± 20 21.5 ± 0.8 1110 ± 210 n/a^(g) n/a^(g) 4.87 ± 0.04 3± 1 n/a

Error bars represent standard deviation for n ≥ 3 separate syntheses.^(b)Estimated using electron micrographs. ^(c)Measured via dynamic lightscattering. ^(d)Nitrogen wt % measured by elemental analysis.^(e)Determined by BET analysis of the nitrogen sorption isotherms (0.05≤ p/p⁰ ≤ 0.20). ^(f)Calculated via BJH analysis on the nitrogenadsorption isotherm (p/p⁰ ≤ 0.60).

Particle sedimentation interfered with DLS measurement.

Pore width could not be calculated from the adsorption isotherm.

indicates data missing or illegible when filed

While significant particle nitrogen content was measured by elementalanalysis, solid-state cross-polarization (¹H/²⁹Si)/magic angle spinning(CP/MAS) nuclear magnetic resonance spectroscopy (NMR) provided evidencefor covalent attachment of AEAP3 to the inorganic TEOS backbone (FIG.3). The Q-band peaks at −94, −103, and −112 ppm were assigned tobackbone Si atoms present as geminal silanol (Q2), lone silanol (Q3),and siloxane (Q4) species, respectively. The T-band, indicative of thebound organosilane (AEAP3), consisted of peaks at −60 and −69 ppm thatwere assigned to T₂ and cross-linked T₃ species, respectively. Theprevalence of cross-linked surface-bound aminosilanes is attributed tothe large water concentration (>20 M) in the reaction mixtures, thatdrives condensation between aminosilanes. For comparison, MCM-41materials produced through post-synthetic surface grafting in anhydroussolvents are primarily bidentate T₂ species and exhibit limitedcross-linking (T₃+T₃′).

NITRIC OXIDE RELEASE: After confirming covalent aminosilane attachment,AEAP3-modified particles were functionalized with N-diazeniumdiolatemoieties by reaction with NO gas at high pressure in the presence ofsodium methoxide. Nitric oxide release was evaluated in real-time viachemiluminescent analysis of the NO-releasing particles in physiologicalbuffer (PBS, pH 7.4) at 37° C. (Table 4). Upon immersion into aqueoussolution the AEAP3/NO MSNs were characterized by a large instantaneousNO flux corresponding to reaction of the proton-labileN-diazeniumdiolate with water to generate NO. Despite large total NOstorage (>0.8μmol/mg) for all four particle systems, total NO storage(p<0.01), NO-release half-lives (p<0.01), and release durations (p=0.02)were unexpectedly diverse. The 1100 nm particles exhibited large NOstorage (1.41 μmol/mg) and rapid release (t½=25.6 mm). Similarly, the 30nm ABAP3/NO particles released their total NO payload rapidly (t½=27.4min) but stored only a fraction of the NO measured for the 1100 nmparticles ([NO]t=0.88 μmol/mg). While the 450 nm MSNs were characterizedwith low NO storage (0.82 μmol/mg), they were associated with thelongest NO-release half-life (88.2 min). Relative to the 1100 nmAEAP3/NO particles, the 150 nm MSNs exhibited comparable NO storage(1.30 μmol/mg) but intermediate NO-release rates (t½=41.9 min).

TABLE 4 Chemiluminescent NO release measurements in physiological buffer(PBS, pH 7.4, 37° C.) from AEAP3/NO MSNs of varying size.^(a)N-Diazeniumdiolate Particle Size [NO]_(max) t_(1/2) t_(d) [NO]_(t)Formation Efficiency (nm) (ppm mg⁻¹)^(b) (min)^(c) (h) (μmol mg⁻¹) (%)30 18.7 ± 2.2 27.4 ± 8.9  12.2 ± 3.0 0.88 ± 0.05 26.6 ± 1.8 150 22.6 ±4.4 40.7 ± 11.0 16.7 ± 1.4 1.30 ± 0.11 30.9 ± 2.7 450  6.6 ± 1.8 88.2 ±10.5 14.0 ± 0.3 0.82 ± 0.08 22.8 ± 2.3 1100 32.8 ± 9.8 25.6 ± 5.0  11.1± 0.7 1.41 ± 0.19 40.7 ± 5.2 ^(a)Error bars represent standard deviationfor n ≥ 3 separate syntheses. ^(b)Maximum instantaneous NOconcentration. ^(c)Half-life of NO release. ^(d)NO-release duration;time required for NO concentrations to reach ≤10 ppb mg⁻¹. ^(e)Total NOrelease. ^(f)Calculated using total NO release and nitrogen wt %determined by elemental analysis (Table 3) according to equationprovided in Supporting Information.

The difference in NO-release kinetics between particle systems was notanticipated, as all particles were functionalized with the sameN-diazeniumdiolate precursor (AEAP3). To shed further light on theseeffects, total NO release from the ABAP3/NO particles were compared tothe degree of nitrogen incorporation measured by elemental analysis(Table 3) to determine N-diazeniumdiolate formation, efficiencies. Asexpected based on the NO release data, the 1100 nm MSNs exhibited thegreatest NO donor formation efficiency (40.7%), far greater than thatreported by others (<27%). The NO donor formation efficiencies for theother three particle sizes were calculated at 23-31%.

The wide range of NO-release kinetics (half-lives 27-88 min) suggestedadditional factors were influencing the NO release. Without being boundby theory, it was hypothesized that the structure and ordering of theparticle pore network may account for these variations, as a linkbetween mesoscopic ordering and diffusion-based drug release has beendemonstrated previously. For example, decreased organization may impedesodium methoxide access to pore-bound secondary amines, hinderingN-diazeniumdiolate. As an extension of the same logic, altered waterdiffusion into the pores would give rise to differences in NO-releasekinetics between AEAP3/NO MSNs of different size. Powder small-angleX-ray scattering (SAXS) was used to assess pore ordering of the bareMSNs.

The SAXS profile for the 1100 nm MSNs (FIG. 4a ) exhibited an intensescattering peak at 0.170 Å⁻¹ (2θ=2.41 o; hkl 100) and two weaker,larger-angle peaks in the scattering profile were assigned to the 110(0.292 Å⁻¹) and 210 (0.339 Å⁻¹) reflections indexed on a hexagonallattice (lattice constant a=43.1±1.5 Å). While the absence ofhigher-order peaks indicated only modest mesoscopic ordering, thescattering profile consisted of the prominent structural lines forMCM-41-type (hexagonal) silica. In contrast to the observed MCM-41structure for the largest particles, the scattering profile for the 30nm MSNs alluded to an alternative mixed pore structure. Analysis of thesmallest particles revealed three scattering peaks: 100 (0.155 Å⁻), 200(0.301 Å⁻¹), and 300 (0.552 Å⁻¹), typical of lamellar (layered) poreordering. The appearance of the 300 reflection represented a high degreeof pore ordering; this peak is seldom observed for lamellar mesoporoussilica. However, the 200 reflection was relatively broad, suggestingthat the pore structure for the 30 nm particles was an intermediateproduct (i.e., between lamellar and hexagonal). The electron micrographsfor the 30 nm MSNs (FIG. 2d ) were in good agreement with the scatteringdate and provided evidence for both pore structures. X-ray scatteringpatterns obtained for the 150 and 450 nm particles were representativeof a greater degree of pore disorder. Other than the 100 line, only abroad peak centered at ˜0.32 Å⁻¹ appeared in both scattering profiles.The absence of the 300 reflection suggests decreased pore organizationfor both particle systems. Indeed, both scattering profiles impliedmesopore arrangements between hexagonal and lamellar structures. Whilepore disorder was not as extensive for 150 nm particles, the skewed 100peak for the 450 nm MSNs was evidence for a more heterogeneous porestructure. In fact, the irregular peak shape was likely thesuperimposition of two separate low order reflections.

Particle x-ray scattering data provided insight into the relationshipbetween MSN pore structure and NO-release kinetics. For the largest 1100nm particles, the ordered hexagonal pore system that was elucidated viaSAXS analysis likely allows for unrestricted pore access by sodiummethoxide and water, resulting in large NO storage and rapid NO release,respectively. The 150 and 450 nm particles were capable of moresustained NO release due to mixed and disordered pore structure. Incontrast, the 30 nm MSNs exhibited rapid NO release resulting from ahighly ordered structure that was largely lamellar in character. Thelower NO storage (0.88 μmol/mg), unaccounted for by the lamellar porestructure, is likely due to poor dispersal of the 30 nm AEAP3 particlesin the N-diazeniumdiolate reaction. Of note, SAXS analysis of theamine-modified particles was also carried out with the results shown inFIG. 8. The scattering profiles of the amine-functionalized particleseach exhibited similar pore ordering to their corresponding bare MSNs,although the peaks were broader and lower in intensity, corresponding topore-filling by the aminosilanes.

ORGANOSILANE MODIFICATION: While aminosilanes are highly reactive withthe silanol groups that populate the surface of silica nanoparticles,they also readily undergo hydrolysis and auto-condensation in aqueousconditions to form new, discrete entities. For this reason, addition oforganosilane directly to the colloidal sol (i.e., particle reactionmixture) generally yields amorphous materials with heterogenousfunctional group distribution. Post-synthetic grafting approaches thusrequire water removal from the reaction mixture to avoid undesirableparticle agglomeration.

In addition to anhydrous conditions, efficient particle modification iscontingent upon successful removal of the pore-resident surfactant priorto reaction with aminosilanes, as the positively charged templatemolecule stabilizes the anionic surface silanols and may impedediffusion of external species into the pores. Others have previouslyexploited the stability of the surfactant CTAB template for selectivederivatization of the outer and inner mesoporous silica surfaces using astep-by-step functionalization approach. In an embodiment, the presentinvention relates to a large degree of particle functionalizationwherein the aminosilanes likely displaced CTAB before undergoing anyauto-condensation. Without being bound by theory, it is postulated thatthis phenomenon might be due to an ion-exchange process between thesurfactant and protonated aminosilanes (FIG. 5). Others have describedion exchange between cationic species (metal ions and aminosilanes,respectively) and the CTAB template as a method for particlemodification. In these cases, the uncalcined (i.e., CTAB-containing)silica was modified in a separate reaction rather than a one-stepprocedure.

In an embodiment, the present invention relates to MSN modification withcationic species, showing that cationic species retain their particlemorphology. Using the 150 nm particle system, the MSNs werefunctionalized with either isobutyl(trimethoxy)silane (BTMS) or(3-mercaptopropyl)trimethoxysilane (MPTMS) at concentrations equal tothose employed for the 150 nm AEAP3 particles. As the colloidal sol isformed under basic conditions, the BTMS alkyl groups remain neutralwhereas a significant fraction of the MPTMS side chains would exist asthe anionic thiolate species (pKa˜10), in both eases preventing ionexchange. 3-aminopropyltriethoxysilane (APTES) was used as a positivecontrol, as APTES is similar in size to BTMS and MPTMS but shouldundergo efficient ion exchange with CTAB due to the presence of a basicprimary amine.

The morphology of the 150 nm APTES, BTMS, and MPTMS particles wasexamined using transmission electron microscopy (FIG. 6). As expected,150 nm particles functionalized with APTES exhibited uniform morphologywith excellent sphericity, consistent with the 150 nm AEAP3 MSNs.Evaluation of aqueous APTES particle suspensions by DLS indicated thatthe monodispersity of the particles (PDI=0.03±0.02) was preserved uponaminosilane modification. In contrast, undesirable silane bridging andparticle agglomeration were evident in electron micrographs of theMPTMS- and BTMS-modified particles. A significant increase in the carbonwt % (measured by elemental analysis; Table 6) for all particle systemsindicated that the silanes were incorporated into the final product. Themorphological differences between particles observed using TEM were dueto reaction with organosilanes. Accurate DLS analysis of the MPTMS andBTMS particle dispersions indicated significant sample polydispersityand agglomeration. While this data does not exclude the possibility ofalternative reaction mechanisms, the particle analyses presented provideclear support of ion exchange reactions between cationic organosilanesand CTAB.

TABLE 5 Physicochemical characterization of 30 nm NO-releasing MSNs as afunction of aminosilane modification.^(a) Particle Characterization NORelease Aminosilane Geometric Z-average Nitrogen [NO]_(t) ModificationSize (nm)^(b) Size (nm)^(c) PDI^(e) wt %^(d) t_(1/2) (min)^(e) t_(d)(h)^(f) (μmol mg⁻¹)^(g) MAP3 37.1 ± 8.3 91.2 ± 8.8 0.16 ± 0.05 3.26 ±0.15 2.2 ± 0.2  1.8 ± 0.4 1.39 ± 0.10 AHAP3 42.3 ± 8.1 131.8 ± 9.4  0.17± 0.04 4.18 ± 0.05 4.7 ± 2.3  5.9 ± 0.2 1.20 ± 0.10 AEAP3 35.7 ± 8.174.1 ± 6.2 0.12 ± 0.06 4.65 ± 0.19 27.4 ± 8.9  12.2 ± 3.0 0.88 ± 0.05DET3 34.5 ± 7.6 83.0 ± 7.6 0.17 ± 0.04 5.60 ± 0.31 47.0 ± 11.9 33.2 ±4.7 1.37 ± 0.19 ^(a)Error bars represent standard deviation for n ≥3separate syntheses. ^(b)Estimated using electron micrographs.^(c)Measured via dynamic light scattering. ^(d)Nitrogen wt % measuredvia elemental analysis. ^(e)Half-life of NO release. ^(f)NO-releaseduration; time required for NO concentrations to reach ≤10 ppb mg⁻¹.^(g)Total NO release.

Table 6 shows characterization of AEAP3-modified 1100 nm mesoporoussilica particles as a function of the reaction aminosilaneconcentration.^(a)

[AEAP3] t_(1/2) t_(d) [NO]_(t) (mM) (min)^(b) (h)^(c) (μmol mg⁻¹)^(d)1.43 14.2 ± 1.6 7.2 ± 1.2 0.56 ± 0.09 2.87 15.8 ± 4.2 7.5 ± 0.3 0.69 ±0.04 5.73 16.8 ± 4.1 9.0 ± 0.5 1.02 ± 0.04 11.47 25.6 ± 5.0 11.1 ± 0.7 1.41 ± 0.19AMINOSILANE MODIFICATION AND NITRIC OXIDE-RELEASE KINETICS: As thestructure of the precursor amine for N-diazeniumdiolate formationinfluences NO-release kinetics from both small molecules and nonporoussilica particles, the NO-release kinetics from the MSNs were alteredusing different organosilanes. The 30 nm particle system wassystematically modified with several aminosilanes, including AHAP3,DET3, and MAP3.

The characterization of the precursor- and NO donor-modified MSNs isprovided in Table 5. Both the geometric size (˜35-43 nm) and PDI (<0.20)of the particles remained approximately constant (p>0.5), indicatingthat the small particle size and monodispersity were preserved duringthe chemical modification regardless of aminosilane type. The measuredhydrodynamic diameter (Z-average size) of each particle system (75-130nm) was dependent on the composition of the aminosilane, but agreed wellwith the corresponding geometric sizes. The nitrogen wt % for each MSNsystem varied expectedly based on the elemental composition of theaminosilane reactant. Particles functionalized with the monoamine MAP3incorporated the least amount of nitrogen (3.26%), while the nitrogen wt% was greatest tor the triamine DET3 modification (5.60%). Intermediatenitrogen content was measured for MSNs with attached AHAP3 (4.18%) andAEAP3 (4.65%), which are diaminosilanes of differing carbon content.

The large degree of aminosilane incorporation translated to excellentparticle NO storage, exceeding 1.00 μmol/mg for all particle systemstested except AEAP3/NO. The lower total NO storage for AEAP3/NO wasexpected based on previous results, as intramolecular hydrogen bondingbetween the side chain amines hinders N-diazeniumdiolate formation.While these interactions are also possible for DET3, the presence of twosecondary amines resulted in greater NO storage. As expected, the MSNNO-release kinetics were markedly different between the four particlesystems (p<0.01). The MAP3/NO and AHAP3/NO particles were characterizedwith rapid initial NO release (t½ of 2.2 and 4.7 min, respectively),while the NO release for the AEAP3/NO and DET3/NO particles was moresustained (t½ of 27.4 and 47.0 min, respectively) as a result ofN-diazeniumdiolate charge stabilization by neighboring protonated amines(Table 5; FIG. 8). The NO-release durations covered ˜2-33 h, renderingthese particles especially useful as NO-delivery vehicles where tuningNO-release kinetics is critical to efficacy.

NO-release payloads, halt-lives, and durations are all somewhatdependent on the particular synthesis (e.g., on the particle size andaminosilane used). However, typical NO storage values for the synthesesdescribed herein are 0.8-1.6 umol/mg (with nothing lower than 0.8). Theparticles using the aminosilane MAPS tended to store more NO (1.5-3.0umol/mg) than using the other aminosilanes. For each size, one can tunethe half-life and duration by the aminosilane identity—all of thesyntheses are amenable to different amine modifications without changingthe size/PDI. Particles modified with MAP3 release for 2-6 h and havehalf-lives of 2-5 min. AEAP3-based particles generally have hall-livesin the range of 30-90 min and durations of 14-18 h regardless ofparticle size. DET3 gives the longest duration of release (33-50 h) withsimilar NO-release half-lives relative to the AEAP3 particles.

It should be noted that others have reported macromolecular NO donorscaffolds with total NO release values exceeding ˜1.5 μmol/mg. Forexample, several porous metal organic frameworks (MOFs) have beendeveloped which with NO storage approaching 1-7 μmol/mg through directadsorption of NO gas. However, NO release from MOFs is generally rapid,restricting their utility to applications in which the NO donor scaffoldis in contact with humidified gas. Both dendrimers and silica particlesmodified with S-nitrosothiol (RSNO) NO donors also exhibit large NOpayloads (2 and 4 μmol/mg, respectively) with NO-release durationsexceeding two days in deoxygenated PBS buffer. Unfortunately, RSNOs areunstable NO donors, readily decomposing to yield NO under multipletriggers (e.g., light, heat, reaction with Cu+ ions or ascorbate).S-nitrosothiol stability is further compromised in the presence ofoxygen, where reaction with NO produces nitrogen trioxide, aRSNO-reactive species that initiates excessively rapid autocatalyticdecomposition. In contrast, N-diazeniumdiolate NO donors alleviate theissue of uncontrolled decomposition, liberating NO at rates dependent onboth the structure of the aminosilane and the solution pH. While poor NOstorage and difficult synthetic procedures have traditionally excludedN-diazeniumdiolate-modified macromolecular NO donors from therapeuticevaluation, the preparation of NO-releasing mesoporous silica particleswas achieved in high yields via ion exchange reactions. Excellent NOstorage and diverse NO-release kinetics from the MSNs were obtained bysimply changing the aminosilane without further synthetic optimization,representing a significant improvement to N-diazeniumdiolate-basedNO-delivery vehicles.

In an embodiment, the present invention has elucidated to some extentthe intricate relationships between pore ordering and NO-releasekinetics. In one variation, the controlled mesophase structure shouldalso provide an additional degree of control in macromolecular NO donordesign. Moreover, the ability to easily modify the MSNs with differentaminosilanes enabled tuning of NO-release kinetics without sacrificingcontrol over either total NO storage or particle size. In an embodiment,the panicles of the present invention should be useful for therapeuticutility. In one variation, the antibacterial action of the smaller 30and 150 nm particles is postulated. The 450 and 1100 nm NO-releasingparticles, while generally unsuitable as antimicrobials due to theirlarge size, are postulated as being useful as dopants for NO-releasingpolymer composites.

EXPERIMENTAL SECTION

MATERIALS: Tetraethylorthosilicate, 3-aminopropyltriethoxysilane,3-mercaptopropyltrimethoxysilane,3-(trimethoxysilylpropyl)diethylenetriamine,N-methylaminopropyltrimethoxysilane,N-(6-aminohexyl)aminopropyltrimethoxysilane, and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane were purchased from Gelest (Morrisville,Pa.) and stored under nitrogen atmosphere. Sodium methoxide (NaOMe; 5.4M in methanol), anhydrous N,N-dimethylformamide (DMF), anhydrousmethanol (MeOH), ethanol (EtOH), aqueous ammonium hydroxide (30 wt %;NH₄OH), concentrated hydrochloric acid and all salts were purchased fromFisher Scientific (Fair Lawn, N.J.). Cetyltrimethylammonium bromide waspurchased from Sigma (St Louis, Mo.). Nitrogen (N₂), argon (Ar), andnitric oxide (NO) calibration (25.87 ppm In nitrogen) gases werepurchased, from Airgas National Welders (Raleigh, N.C.). Pure NO gas waspurchased from Praxair (Danbury, Conn.). Water was purified using aMillipore Milli-Q UV Gradient A10 system (Bedford, Mass.) to aresistivity of 18.2 MΩ·cm and a total organic content of <10 ppb. Unlessspecified, all chemicals were used as received without furtherpurification.

NANOPARTICLE SYNTHESIS: Tetraethylorthosilicate was added as a bolus toa stirred solution of water, EtOH, NH₄OH, and CTAB and allowed to reactfor 2 h. For the 30, 150, and 450 nm syntheses, 2.500 mL TEOS in EtOH(0.88, 1.06, and 1.33 M, respectively) was added to the reactionmixture, whereas 1.395 mL concentrated TEOS was used for the synthesisof the larger 1100 nm particles. Synthesis conditions for the MSNs areprovided in Table 1. In all cases, reaction solutions appeared turbidwithin 15 min of silane introduction. Following particle formation,additional organosilane (AEAP3, AHAP3, APTES, BTMS, MAP3, MPTMS, orDET3) was introduced directly to the colloidal sol dropwise for 5 minusing a Kent Scientific Genie Plus syringe pump (Torrington, Conn.).Elemental analysis of the 150 nm APTES, BTMS, and MPTMS particles areshown in table 7. The reaction was then aged overnight (˜18 h) withstirring. Unless specified, an optimized TEOS:organosilane molar ratioof 1.56:1.00 was used. Following functionalization, particles werecollected by centrifugation (6540 g, 4° C., 15 min), washed three timeswith EtOH, and dried under vacuum. For both the 30 and 150 nm particles,EtOH (one volume per two volumes of the reaction mixture) was added tothe sol to induce particle flocculation during the collection procedureand enhance the overall yield. Bare MSNs were synthesized and collectedsimilarly but without the organosilane functionalization step.

TABLE 7 Elemental analysis of 150 nm APTES, BTMS, and MPTMS particles.Silane Modification Carbon wt % Hydrogen wt % Nitrogen wt % APTES 13.214.08 4.49 BTMS 23.16 4.79 0.16 MPTMS 25.58 4.84 0.88

Following MSN synthesis, residual CTAB was removed by ion exchange withhydrochloric acid (HCl). Particles (˜200 mg) were suspended in 30 ml 10%v/v HCl in EtOH, agitated in an ultrasonicator bath for 30 min, andcollected by centrifugation (6540 g, 4° C. 15 min). This process wasrepeated three times to ensure complete CTAB removal, followed by twoadditional EtOH washes. The particles were dried under vacuum to yieldthe pure nanoparticles. Typical yields for the amine-modified 30, 150,450, and 1100 nm MSNs were 150, 175, 275, and 650 mg, respectively.

MESOPOROUS SILICA NANOPARTICLE CHARACTERIZATION: Particle morphology wascharacterized using a JEOL 2010F transmission electron microscope(Peabody, Mass.). Particles were suspended in MeOH at 1 mg/mL via briefagitation with an ultrasonicator. Subsequently, 5 μL of the resultingdispersion was cast onto a Formvar-coated copper grid (Ted Fella, Inc.;Redding, Calif.). The geometric size distribution of the particles wasestimated from the electron micrographs using ImageJ software (Bethesda,Md.). The solution-phase behavior of the nanoparticles in water wasinvestigated using dynamic light scattering (Malvern Zetasizer Nano-ZS;Westborough, Mass.) to determine MSN hydrodynamic diameter (Z-averagesize) and polydispersity index. Aqueous colloidal nanoparticlesuspensions were prepared by dispersing particles at a concentration of0.5 mg/mL using probe sonication at 7 W for 45 s using a Misonix S-4000ultrasonicator (Farmingdale, N.Y.). Nitrogen sorption isotherms werecollected on a Micromeritics Tristar II 3020 surface area and porosityanalyzer (Norcross, Ga.). Samples were dried under a stream of N₂ gas at110° C. overnight and then degassed for 2 h prior to analysis.Brunauer-Emmett-Teller (BET) analysis of physisorption data was used tocalculate MSN specific surface area for p/p⁰ values of 0.05-0.20. Poresize analysis using the adsorption branch of the sorption isotherm(0.05<p/p⁰<0.60) was using the Barrett-Joyner-Halenda (BJH) method. Dataobtained at relative pressures >0.60 p/p⁰ were not considered for poresize determination as nitrogen capillary condensation occurred in theinter-particle volumes for the 30 nm and 150 nm particles, inflating thecalculated pore width. Pore structure/ordering information was obtainedby small-angle X-ray scattering analysis of the dry MSN powder. The CuKα line (1.54 Å) was used as the source radiation and scatteringprofiles were collected on a SAXSLab Ganesha point collimated pinholesystem equipped with a moveable Dectris Pilatus 300K 2-dimensionalsingle-photon-counting detector (Northampton, Mass.). Scattering vector(q) calibration was accomplished using the 1st-order ring for silverbehenate, and data was collected for q-values of 0.005-0.724 Å⁻¹.Covalent incorporation of aminosilanes into the MSN backbone wasconfirmed via solid-state cross-polarization/magic angle spinning(CP/MAS) ²⁹Si nuclear magnetic resonance spectroscopy using a Bruker DMX360 wide-bore spectrometer at a resonance frequency of 71.548 Hz.Samples were carefully ground in a mortar and pestle, packed into a 4 mmZrO₂ rotor, and spun at 10 kHz. All chemical shifts were determinedrelative to an external tetramethylsilane standard. Elemental analysiswas used to quantify the nitrogen weight percent of particles before andafter functionalization with secondary amine-containing silanes using aPerk in Elmer 2400 CHNS/O analyzer (Waltham, Mass.) operated in CHNmode.

N-DIAZENIUMDIOLATE MODIFICATION AND NITRIC OXIDE RELEASE MEASUREMENTS:The aminosilane-modified MSNs (˜15 mg) were suspended in 9:1 DMF:MeOH at5 mg/mL in a glass vial and dispersed by ultrasonication for 20 min.After forming a homogeneous particle dispersion, NaOMe (5.4 M in MeOH;9.0 μmol per mg MSN) was added to the solution and mixed. TheMSN-containing vials were equipped with stir bars, placed in a stainlesssteel reaction bottle (Parr Instrument Co.; Moline, Ill.), and connectedto an in-house NO reactor. The Parr bottle was Hushed six times (threerapid, three 10 min) with 8 bar Ar gas to remove atmospheric oxygen andminimize the formation of NO byproducts. The vessel was subsequentlypressurized with 10 bar NO gas and allowed to react for 72 h. Of note,the NO gas used for N-diazeniumdiolate formation was purified over solidpotassium hydroxide for at least 4 h prior to reaction. After 72 h, theParr bottle was vented and the vessel was flushed six more times (threeshort, three 10 min) to remove unreacted NO. The particles were againcollected by centrifugation (6540 g, 4° C., 15 min), washed three timeswith EtOH, and dried under vacuum for 1-2 h. The resultingN-diazeniumdiolate-modified particles were stored in a vacuum-sealed bagat −20° C. until further use.

Nitric oxide release measurements were carried out using a Sievers 280iNO analyzer (Boulder, Colo.). Generation of NO from the proton-labileN-diazeniumdiolate NO donors was detected indirectly viachemiluminescence from excited, state nitrogen dioxide formed upon thereaction of NO with ozone. The NOA was calibrated using a two-pointlinear calibration; air passed through a Sievers NO zero filter servedas the blank value and 25.87 ppm NO in N₂ was used as the secondcalibration point. Particles (˜1 mg) were added to the NOA sample flaskcontaining 30 mL deoxygenated phosphate buffered saline (PBS, 0.010 M,pH 7.41) at 37° C. A stream of N₂ gas (80 mL/min) was continuouslybubbled through solution to carry liberated NO to the analyzer.Supplemental nitrogen flow was provided to the flask to match theinstrument collection rate of 200 mL/min. Instantaneous NOconcentrations were measured, at a sampling frequency of 1 Hz, providingnear real-time information regarding MSN NO-release kinetics. The NOmeasurements were terminated when NO release from the particles wasbelow 10 ppb/mg.

STATISTICAL ANALYSIS: One-way Analysis of Variance was used for multiplecomparisons of MSN physicochemical properties (e.g., surface area, poresize, NO-release total amounts and kinetics) with provided p-values.Individual comparisons were carried out using a two-tailed Student'st-test with α=0.05 considered as the threshold for statisticalsignificance.

In one embodiment, the present invention relates to ion exchange betweencationic organosilanes and alkyltrimethylammonium SDAs(structure-directing agents), which represents a new MSN (mesoporoussilica nanoparticle) functionalization approach.

In one embodiment the organosilanes of the present invention areorganoaminosilanes and they are used in cationic ion exchange and theyare represented by the compounds of Formula I

wherein z is 0, 1, or 2; and R₁ is

wherein R₂ and R₃ are each independently H, CH₃, (CH₂)₂₋₅NH₂, or(CH₂)₂₋₅NH(CH₂)₂₋₅NH₂.

In an embodiment, the organoaminosilanes are one or more of:

aminosilane N-(2-aminoethyl)-3-aminopropyltrimethoxysilane:,

N-(6-aminohexyl)aminopropyl trimethoxysilane,

(3-Aminopropyl)triethoxysilane,

N-methylaminopropyltrimethoxysilane,

(3-trimethoxysilyl-propyl)diethylenetriamine or combinations thereof.

In an embodiment the present invention allows one to vary reactionconditions so as to get appropriately sized mesoporous silicananoparticles. In an embodiment, the syntheses of the present inventionallow controlled morphology wherein one can generate mesoporous silicananoparticles with a useful surface area in the range of about 600-1400m²/g. Also, the synthetic conditions of the present invention allow oneto attain particles with extremely ordered pore systems (for exampleeither rods, or 2D hexagonal systems) to intermediate and moredisordered pore systems (more typically). In an embodiment, the presentinvention allows one to produce particles with pore of a size of about15-25 Å and with pore volumes of about 0.4-1.0 cm³/g. In one embodiment,a synthesis of the present invention produces rods with much largerordered pores in a size range of about 85-95 Å (for example, about 88 Å)ordered pores (2D hexagonal) with greater pore volumes of about 1.2-1.5cm³/g. Of note, generally pore volume correlates with pore size, solarger pores generally also yield greater pore volumes.

In an embodiment, the use of surfactant templated synthesis ofmesoporous silica relies on silica formation around micelles. In avariation, using this synthetic method, one introduces 2-10 nm pores(e.g., cylindrical) into silica particles. In one variation, theinterior (pore) surface can be chemically modified. For mesoporoussilica, the surface area may be on the order of about 1,200 m²/g,whereas it is on the order of less than about 200 m²/g for nonporoussilica. The reported surface area measurements for the four particlesystems described are 1170-1290 m²/g. Reported surface area values arein the range of 700-1600 m²/g, with 800-1000 m²/g being most common.

In an embodiment, a surfactant removal step is performed (e.g.,agitation in ethanol/hydrochloric acid).

Using the ion exchange methods discussed herein, one is able to achievea combination of features that cannot be achieved by using themethodologies of the prior art. For example, table 8 shows a comparisonof the various methods that can be used to generate NO-releasing silicasystems.

For example, and as shown in Table 8, the ion exchange methodology asdescribed herein generates good NO storage capabilities, with a relativelong NO-Release half life, wherein one can modulate size control.Moreover, there are few synthesis concerns as a one-pot reaction can beemployed to generate these good NO-releasing silica systems. Because,the synthetic method is water-based, it is not subject to sensitivity tohumidity unlike some of the other methodologies.

TABLE 8 comparison of different methods of generating NO-ReleasingSilica Systems NO Storage NO-Release Size Synthesis Synthesis Porosity(μmol mg⁻¹) Half-Life (h) Control? Concerns Reference(s) Co- Nonporous0.05-0.68 0.7-6.0 No None J. Am. Chem. Soc. 2007, 4612-4619 condensationCo- Nonporous 0.22-0.39 0.1-2.0 No None J. Dent. Res. 2014, 1089-1094condensation J. Dent. Res. 2015, 1092-1098 Grafting Nonporous 0.24-0.700.1-0.8 Yes Water sensitive ACS Appl. Mater. Interfaces 2013, 9322-9329Small 2013, 2189-2198 Grafting Nonporous 0.26-0.53 ~0.9 h Yes WaterSensitive J. Am. Chem. Soc. 2003, 5015-5024 Co- Nonporous 0.27-0.30 Notreported No None Biomacromolecules 2012, 3334-3342 condensationMicroemulsion Nonporous 1.00-1.49 ~2-3 h Yes 500 mL alcohol ACS Nano2011, 7235-7244 (hybrid) solvent 10 mg yield 2 d synthesis Ion ExchangeMesoporous 0.80-2.90 0.2-5.4 Yes None ACS Appl. Mater. Interfaces 2016,2220-2231

In an embodiment, the present invention relates to a method of producingNO-releasing mesoporous silica particles, wherein said method comprises:generating a mesoporous silica particle by reacting tetraalkoysilanes oralkoxysilanes with a cetyltrimethylammonium halide to generate acetyltrimethylammonium ion.

In a variation, the present invention relates to reactingtetraethylorthosilicate with a cetyltrimethylammonium halide to generatea cetyltrimethylammonium ion; exchanging via an ion exchange reactionthe cetyltrimethylammonium ion for an organosilane molecule.

Other tetraalkoysilanes or alkoxysilanes that may be used in the presentinvention include tetraethylorthosilicate, or methyltrimethoxysilane,ethyltrimethoxysilane, propyltrimethoxysilane,isopropyltrimethoxysilane, butyltrimethoxysilane,isobutyltrimethoxysilane, and t-butyltrimethoxysilane (i.e.,alkoxysilanes with a single alkyl chain side group).

In one variation, the cetyltrimethylammonium halide may becetyltrimethylammonium bromide.

In a variation, the ion exchange reaction is a cation exchange reaction.

In an embodiment, the organosilane is an organoaminosilane and theorganoaminosilane may be a compound of Formula I:

wherein z is 0, 1, or 2; and R₁ is

and wherein R₂ and R₃ are each independently H, CH₃, (CH₂)₂₋₅NH₂, or(CH₂)₂₋₅NH(CH₂)₂₋₅NH₂.

In one variation, the organoaminosilane may be one or more of:

aminosilane N-(2-aminoethyl)-3-aminopropyltrimethoxysilane,

N-(6-aminohexyl)aminopropyl trimethoxysilane,

(3-Aminopropyl)triethoxysilane, N-methylaminopropyltrimethoxysilane, and

(3-trimethoxysilyl-propyl)diethylenetriamine or combinations thereof.

In one variation of the method, the method further comprises chargingthe NO-releasing mesoporous silica particles with NO.

In an embodiment, the NO-releasing mesoporous silica particles have manyadvantages such as:

-   -   a) produce consistent NO-releasing mesoporous silica particles        morphology regardless of aminosilane incorporation b) allow for        a one-pot synthesis of amine-functionalized NO-releasing        mesoporous silica particles c) produce NO-releasing mesoporous        silica particles that are insensitive to ambient humidity and d)        provide NO-releasing mesoporous silica particles wherein        aminosilane incorporation is reproducible.

In one variation, the method allows one to realize all of theadvantages. In one variation of the method, the generation of mesoporoussilica particles can be procured in a one-pot reaction process.

In one variation, the size of the NO-releasing mesoporous silicaparticles is between about 30 nm and 1100 nm. In one variation, theNO-releasing mesoporous silica particles are substantially monodispersein size. To determine the meaning of substantially monodisperse in size,one should look to the error and/or standard deviation parameters asenumerated above, wherein standard statistical methods are used todetermine the extent of deviation from the various sized NO-releasingmesoporous silica particles that have been made. In one embodimentsubstantially monodisperse in size means that the appropriately numberedNO-releasing mesoporous silica particles are within one standarddeviation unit in a normal distribution curve (e.g., using standardstatistical methods).

In one variation, the method relates to varying a combination of aconcentration of the cetyltrimethylammonium halide and a temperature ofa reaction to generate the NO-releasing mesoporous silica particles thatare substantially monodisperse in size. The reaction conditions (e.g.,concentration and temperature) predominantly determine the sizeNO-releasing mesoporous silica particles that are generated.

In one variation, the NO-releasing mesoporous silica particles are of asize that is one of about 30 nm, 150 nm, 350 nm and/or 1100 nm.

The present invention is not just related to methods but also toNO-releasing mesoporous silica particles that have special properties.For example, in one embodiment, the NO-releasing mesoporous silicaparticles can be charged with NO at a concentration of at least about0.4 μmol/mg and release NO with a half-life for release of the NO thatis no less than about 2 minutes or alternatively, 10 minutes, oralternatively, about 15 minutes, or alternatively, about 20 minutes, oralternatively, about 25 minutes. In a variation, the NO-releasingmesoporous silica particles are substantially monodisperse in size.

In one variation, the NO-releasing mesoporous silica particles are of asize that is one of about 30 nm, 150 nm, 350 nm and/or 1100 nm.

The present invention also relates to the generation of NO-releasingmesoporous silica particles by a process that comprises generating amesoporous silica particle by reacting tetraethylorthosilicate with acetyltrimethylammonium halide to generate a cetyltrimethylammonium ion;and exchanging via an ion exchange reaction the cetyltrimethylammoniumion for an organosilane molecule.

In one variation, the organosilane is a compound of Formula I:

wherein z is 0, 1, or 2; and R₁ is

and wherein R₂ and R₃ are each independently H, CH₃, (CH₂)₂₋₅NH₂, or(CH₂)₂₋₅NH(CH₂)₂₋₅NH₂.

In a variation, the organosilane may be one or more of:

aminosilane N-(2-aminoethyl)-3-aminopropyltrimethoxysilane,N-(6-aminohexyl)aminopropyl trimethoxysilane,

(3-Aminopropyl)triethoxysilane, N-methylaminopropyltrimethoxysilane, and

(3-trimethoxysilyl-propyl)diethylenetriamine or combinations thereof.

It should be understood that the present invention is not to be limitedby the above description. Modifications can be made to the above withoutdeparting from the spirit and scope of the invention. It is contemplatedand therefore within the scope of the present invention that any featurethat is described above can be combined with any other feature that isdescribed above (even if those features are not described together).Moreover, it should be understood that the present inventioncontemplates and it is therefore within the scope of the invention thatany step, element or feature can be added and/or omitted in the methodsto obtain the NO-releasing mesoporous silica nanoparticles of thepresent invention. In any event, the scope of protection to be affordedis to be determined by the claims which follow and the breadth ofinterpretation which the law allows.

We claim:
 1. A method of producing NO-releasing mesoporous silicaparticles, wherein said method comprises: generating a mesoporous silicaparticle by reacting a tetraalkoxysilane or alkoxysilane with aalkylammonium surfactant to generate a alkylammonium surfactant ion;exchanging via an ion exchange reaction the alkylammonium surfactant ionfor an organosilane molecule.
 2. The method of claim 1, wherein thetetraalkoxysilane is tetraethylorthosilicate and the alkoxysilane is oneor more of methyltrimethoxysilane, ethyltrimethoxysilane, orisobutyltrimethoxysilane and the alkylammonium surfactant iscetyltrimethylammonium bromide.
 3. The method of claim 2, wherein thecetyltrimethylammonium halide is cetyltrimethylammonium bromide.
 4. Themethod of claim 3, wherein the ion exchange reaction is a cationexchange reaction.
 5. The method of claim 4, wherein the organosilane isan organoaminosilane.
 6. The method of claim 5, wherein theorganoaminosilane is a compound of Formula I:

wherein z is 0, 1, or 2; and R₁ is

wherein R₂ and R₃ are each independently H, CH₃, (CH₂)₂₋₅NH₂, or(CH₂)₂₋₅NH(CH₂)₂₋₅NH₂.
 7. The method of claim 5, wherein theorganoaminosilane is one or more of: aminosilaneN-(2-aminoethyl)-3-aminopropyltrimethoxysilane,N-(6-aminohexyl)aminopropyl trimethoxysilane,(3-Aminopropyl)triethoxysilane, N-methylaminopropyltrimethoxysilane, and(3-trimethoxysilyl-propyl)diethylenetriamine or combinations thereof. 8.The method of claim 1, wherein the method further comprises charging theNO-releasing mesoporous silica particles with NO.
 9. The method of claim1, wherein the method comprises at least one of the followingadvantages: b) produces consistent NO-releasing mesoporous silicaparticles morphology regardless of aminosilane incorporation b) allowsfor a one-pot synthesis of amine-functionalized NO-releasing mesoporoussilica particles c) produces NO-releasing mesoporous silica particlesthat are insensitive to ambient humidity and d) provides NO-releasingmesoporous silica particles wherein aminosilane incorporation isreproducible.
 10. The method of claim 9, wherein, the method comprisesall of the advantages.
 11. The method of claim 1, wherein the methodcomprises a one-pot reaction process.
 12. The method of claim 1, whereinthe size of the NO-releasing mesoporous silica particles is betweenabout 30 nm and 1100 nm.
 13. The method of claim 9, wherein theNO-releasing mesoporous silica particles are substantially monodispersein size.
 14. The method of claim 13, wherein a combination of aconcentration of the cetyltrimethylammonium halide and a temperature ofa reaction generates the NO-releasing mesoporous silica particles thatare substantially monodisperse in size.
 15. The method of claim 14,wherein the NO-releasing mesoporous silica particles are of a size thatis one of about 30 nm, 150 nm, 350 nm and/or 1100 nm.
 16. The method ofclaim 1, wherein the alkylammonium surfactant is: a. Linearalkylammonium salts with molecular structures [C_(n)H_(2n+1)(CH₃)₃N]⁺Br⁻ or [C_(n)H_(2n+1)(C₂H₅)₃N]⁺ Br⁻ where n=8, 10, 12, 14, 16, 18, 20,22. b. Germinal salts with general molecular structures[C_(n)H_(2n+1)(CH₃)₂N—(CH₂)₈—N(CH₃)₂—C_(m)H_(2m+1)]⁺ Br⁻ where n=m=12,14, 16, 18, 20, 22 and s=2−12. c. Divalent surfactants with generalmolecular structures [C_(n)H_(2n+1)(CH₃)₂N—C_(m)H_(2m+1)(CH₃)₃N]²⁺ wheren+m=8, 10, 12, 14, 16, 18, 20,
 22. 17. NO-releasing mesoporous silicaparticles, wherein said NO-releasing mesoporous silica particles arecharged with NO at a concentration of at least about 0.4 μmol/mg andrelease NO with a half-life for release of the NO that is no less thanabout 2 minutes.
 18. The NO-releasing mesoporous silica particles ofclaim 17, wherein the mesoporous silica particles are substantiallymonodisperse in size.
 19. The NO-releasing mesoporous silica particlesof claim 18, wherein the mesoporous silica particles are of a size thatis one of about 30 nm, 150 nm, 350 nm and/or 1100 nm.
 20. TheNO-releasing mesoporous silica particles of claim 19, wherein theNO-releasing mesoporous silica particles are made by a process thatcomprises generating a mesoporous silica particle by reactingtetraethylorthosilicate with a cetyltrimethylammonium halide to generatea cetyltrimethylammonium ion; and exchanging via an ion exchangereaction the cetyltrimethylammonium ion for an organosilane molecule.21. NO-releasing mesoporous silica particles of claim 20, wherein theorganosilane is a compound of Formula I:

wherein z is 0, 1, or 2; and R₁ is

wherein R₂ and R₃ are each independently H, CH₃, (CH₂)₂₋₅NH₂, or(CH₂)₂₋₅NH(CH₂)₂₋₅NH₂.
 22. The NO-releasing mesoporous silica particlesof claim 21, wherein the organosilane is one or more of: aminosilaneN-(2-aminoethyl)-3-aminopropyltrimethoxysilane,N-(6-aminohexyl)aminopropyl trimethoxysilane,(3-Aminopropyl)triethoxysilane, N-methylaminopropyltrimethoxysilane, and(3-trimethoxysilyl-propyl)diethylenetriamine or combinations thereof.23. NO-releasing mesoporous silica particles of claim 17, wherein saidNO-releasing mesoporous silica particles have a half-life for release ofthe NO that is no less than about 25 minutes.